Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 93/13809 212 i 7 ~ ~i PC~/EP93/0002X
I M P R 0 V E M E N ~ S I N C 0 N T R A S T A ~ E N T S ,
C 0 N S I S T I N G 0 F G A L A C T 0 S E P A R ~ I C L E S .
This invention relates to novel contrast agents,
more particularly to ne~ microparticulate contrast
agents of use in diagnostic imaging.
It is well known that ultrasonic imaging comprises
a potentially valuable diagnostic tool, for example ir.
studies of the vascular system, particularly in
cardiograph~, and of tissue microvasculature. ~ variet,
of contras agen~s has been proposed to enhancs the
acoustic images so obtaineà, in_ludin~ suspensions of
solid par,icles, emulsified liquid droplets, g~s
microbubbles and encapsulated gases or liquids. It is
generall~ accepted that low density con~rast agents
which are ea ily compressible are particularlv efficient
in terms of the acoustic backscatter they generate, and
considerable interest has therefore been shown in the
preparation of gas-containing and gas-generating
systems.
Initial studies involving rree gas microbubbles
generated in vivo b~ intracardiac injection of
physiologicall~ acceptable substances have demonstrated
the potential efficiency of such bubbles as contrast
agents in echocardiography; such techniques are severel~
limited in practice, ho~ever, by the short life_ime of
the free bubbles. Interest has accordin~ly been shown
in methods of generating and/or stabilising gas
microbubbles for echocardiography and other ultrasonic
studies, for example using emulsiliel-s, oils, thickeners
or suyars.
Techniques involvin~ the use of sugars in
ultrasound contrast a~en~s are described in, for
e~ample, US~ ,6~ , U~ ;`,O;~ and VS-.~-
~,65/,75', which disclose the use c- pa-ticulat~ solids
ha~.in-1 a plul~ali~. o~ g2c~-Iille~ O~ an~. preTerabl~
0 93/13809
also a pluralit~r of nuclei for ~ilcrobubcl~ formG~i~r..
EP-A-0123235 and EP-A-0122~24 suggest ul~rasoun_
contrast agents consisting of surfactant-coated c~
surfactant-containing qas-containing microparticles
which may include a variety of sugars. DE-A-3c;3~705
proposes the use of suspensions containing
microparticles of mixtures of at least one C~2 fatty
acid ~ith at least one non-surface active substance,
including sugars such as cyclodextrins, monosaccharides,
disaccharides or trisaccharides, as well as oth--
polyols and inorganic and organic salts.
Gas-containing contrast media are also }~no:.n IO ~e
effective in magnetic resonance (MR? imagin~, e.c;. as
susceptibilit~ contras- agents ~.hich ~:il' ac~ c re~uc~
MR signal intensity. O~gen-containing contras- media
also represent potentially useful paramagnetic 1-.
contrast agents.
Furthermore, in the field o X-ray imaging it has
been observed that gases such as carbon dioxide mav be
used as negative oral contrast agents.
A disadvantage of many existing gas-containing~gas-
generating particulate contrast agents such as the
sugar-based agents discussed above is their relative
lack of stability in vivo. This is a particula- proble~
in appl~cations such as echocardiography, where there is
a need for improved contrast agents combining sufficient
stability and small microbubble size (t~pically less
than about 10 ~m, preferably less than about 7 um) to
permit passage through the pulmonary capillary bed and
so allow enhanced visualisation of the left side of the
heart, preferably for more than one passage of
circulation. There is accol-clingly cl need for contrast
agents wllich generc~te microbubble s~stemC e~:hibi~incl
good stability while stilL providin~ an effectiv* level
3~ of contrast efficienc~.
The present invention is based on our findin~ tha
contrast agentC, co~prising microparticles of a ~ter-
~:O9~/13809 7 ~; 7 7 ~ ~ Pcr/EPg~/o~ 2x
soluble carboh~dra~e admixe~ ~.ith a substantiaproportion (e.c3. at least lO~ ~:/;. relative to the
overall cor~position) of a less ~.ater-soluble non-surface
active material may be used to generate microbubble
systems exhibiting useful levels of contrast effect
and/or stabilit~. In the ultrasound field this may be
demonstrated b~, for example, in ~itro measurements o~
initial attenua~ion levels and the hal lives of the
attenuative effect; a useful indication of the combined
effect of these properties is the integral obtained b~
determinin~3 the area under the curve of a plo Or
attenua~ion agains~ tim~.
Tnu.~, accGrdina to one aspec~ o~ the presen~
inventior, there ar~ pro~ided contras- agen~s com~-isin
wate,-soluble microbubble-generating carbo~ydratc
microparticles in admixture ~ith at least lO~
relative to the overall co~position of a non-s~-face
active material which is less water-soluble than the
said carbohydrale.
The microparticulate carbohydrate ray for example
be selected from he~oses such as glucose, fructose or
galac~ose; disaccharides such as sucrose, lactose o-
maltose; pentoses such as arabinose, xylose or ribose;
and ~ - anc~ ~- c~.clode~lrins; the term "carDohydrate"
as used herein is also intended to embrace sugar
alcohols, e.g. alditols such as ~annitol or sorbitol.
Microparticles of the above carbohydrates ~ill normall~
have gas present as an inclusion in the voids of their
crystal structure and/or adhered to their surface, which
gas may generate microbubbles when, for e~ample, the
microparticles are suspende~l or dissolved in an
injectable carrier liquic~, for e~ample ~ater fol^
in~ection, an aquecl1s so~ution of o1le or more inoraa1lic
salts (e.~. F?11~siolcc1ic.11 s~ o~- C-l Dh~siolc~ic~l
3~ buffer solutio;~ n aquecu~ solution c~` a
monosaccharide (e~c1. c1luccse or c~alactose) or
disaccharide (e.c:. lactose,, o. an aqueous sclu~ion C?l` a
~'093/13X09 PCT/EP93/0002X
2 1~ 7~
physiologically tolerable monohydric or polyh~dric
alcohol (e.g. ethanol, propanol, isopropanol, ethylene
glycol, propylene glycol, glycerine or polyethylene
glycol).
In addition to or alternatively to air, any
biocompatible gas may be employed in the contrast agents
of the invention, for example nitroc3en, oxygen,
hydrogen, nitrous oxide, carbon dio~:ide, helium, argon,
sulphur hexafluoride and low molecular weight optionally
fluorinated hydrocarbons such as methane, ac~.tvlene or
carbon tetrafluoride. The term "gas" as used herein
includes any substance in the gaseous f`or~- a~ ~' C. Tne
gas ma~- ~e contained in the con~ras~ ac~en_ in such a ~a~
that before use the produc- is non-con~ra~ c.i~v~nc3 bu-
17 becomes effective on administration, e.c3. as a resul~ ofthe gas forming microbubbles as the soluble carbohydrate
matri~: dissolves.
Additionally or alternatively the carbohydrate may
incorporate one or more gas precursors, including
car~onates and bicarbonates (e.g. sodium or ammonium
bicarbonate) and a~inomalona~e esters.
The non-surface active material may, for e~ample,
be a high molecular weight polysaccharide such as starch
or de~:tran ~hich exhibits lower ~ate~- solubilit\ than
the micro~ubble-generatinc3 carbohydrate; a non-
amphiphilic lipid such as a fatt~ acic~ ester or steroid
acid este~^; a fixed oil, fa_ or ~a~:, ror e~:ample a
partially hydrogenated vegetable oil such as cottonseed
oil or sovabean oil or a mixture thereof. a
triglyceride, for example a cj~lvceryl ester of a hiclh
molecular ~eic3ht (e.g. C~ ) ali~l1atlc clCid, s~ch as
glyceryl trila~late c~l- c~ c~ryl tLi~ ri~tate; a ~!ax, fOI`
exampl~ a~ c,1rnc~ r.i~:t~ of ~ c~
non-a~phiphilic lipi(1L;. ~lternativ~ o~- ac~clitionall~ a
3~ substantiall~r completel~ ~ater-ill;o~u~le matel-ia~, e.c,.
an inorc3anic material such as silica o~- an iron o~ide
such ~.~.lc1n~ti~i? (~e~ r.. ~
W O 9~/13809 ~ 7 ~ PC-r/EP93/00()2X
,,
~ s indicate~ above, the non-surface actl e mat~riai
will normally be present in an amount of a. least 10-
w/~, for example 20-80~ W/~, advantayeousl~ 25-7
relative to the overall composition. The
microparticulate carbohydrate and non-surface active
material may conveniently De employed in substantiall~
identical amounts by weight.
If desired, the contrast agents according to the
invention may also contain small amounts (e.g. 0.01-5.0
0 W/t~!, preferablv 0.1-2.0% w/~) of raterials such as
amphiphilic lipids in order to modif~ the solubilit~
properties of the microparticles. ~mphiphilic lipic~
~hich r,a~ be used for this purpose inclucie fa.~. aci_s
and salts (~.c,. al}:ali m~eta' sal~s) ~hereo., s~e~oi~
acids, s~erols, phospholipids and gl~colipids. The
amphiphilic lipid may, fo- example, be a high r,olecular
weight (e-g- C1050) straight chaln salurated or
unsatura~ed aliphatic acid, such as capric, palr~itic,
hexadecanedioic, stearic, linolenic, behenic,
docosanedioic or melissic acid; an aral~anoic acid, e.g.
a phenyl lo~.~er al~anoic acid such as 2-phenylbutyric
acid, a cholanic acid such as 5~-cholanic acid; salts of
any of the forec~oing acids; a high molecular ~!eight
(e.g. C1~sn) straight chain aliphatic alcohol such as
stearyl alcohol or cetyl alcohol; a glyceride, for
example a glyceryl ester of a high molecular weight
(e.~. C1os~) aliphatic acid, such as glyceryl
monolaurate; cholesterol; a phospholipid such as
phosphatidyl choline or dioleoylphosphatid~l
ethanolamine; or a mi~ture thereof.
The contrast a~ents of the invention may be use~l in
a variety of clia(7nostic ima~iTl~ tecl~ni~ues, includi~
ultrasouncl, MR and ~-r<l~ ima(1in~l. 'I`heiT use in
diagnostic ultrasonic ima~in~J an~ in Ml~ ima~in~, e.c~. ac
susceptibility contrast a~ents, constitute pl-eferI-e~
features of the invention.
The contras' a~ents of` 'he in~entioJl ma~ b~
W093/13809 2. ~ 2 i 7 V ~ PCT/EP93/()~()2X
prepared by any convenient metho~ ~hich ieads t~
physical admixture cf the carbohydrate and the less
water-soluble non-surface active material and tc
production of microparticles of the desired size. Thus,
S for example, the contrast agents may be prepared simply
by micronising a mixture of the two components, for
example using conventional techniques such as grinding
or milling. Alternatively the two components
(optionally together with any further component cr
components such as the above-described lipids) mav be
mixed in solution, e.g. by mi~:ing an aqueous soiurion of
the carboh~drate an~ a solution OL the less ~ater-
soluble non-surface active material in water or a ~ater-
miscible organic solven~_ as appropriate (togethe- ;~itt:
any desired addi~ive, e.~. a lipid dissolved in a v:a~er-
miscible organic solvent, for example a lower al}:anol
such as ethanol), the solvent or solvents thereafter
being removed (e.g. by evaporation under reduced
pressure~ to yield a mixture of the desired components
which is thereafter micronised to yield the desired
microparticles. It will be appreciated that all
processing operations should be effected unde~ sterile -~
conditions.
In general micronisation ma~ be effected using
conventional techniques such as grinding or milling.
Ball-milling has been found to be particularl~
advantageous, permittin~ the preparation ol
microparticles in the form of aggregates (for e~ample
having an aggregate size of ~0-125 micrometres, such as
30 30-50 micrometres) of particles having a particle si2e
of, for e~ample, 1-50 micrometres, such as 1-1~
micrometres. Such aggregates will tend to contain a
substantial volume ot air adsorbed on their SUl'~aCe~ atlt~
entrainec~ oids ~ ch ac interl-article ca~itiec Ol at
3~ grain bounda~ s bet~een the cr~stallites. The ~rticle
size ma~, for e~ample, be selected to be substan~iall
commens~rate ~.~ith tile c~esirec~ microbu~ble ~i e. n
~093/l3809 21 2 7 q ~ ~ PCT/EP93/0~)02~
ultrasonic applications such as echocardiography, in
order to permit free passage through the pulmonary
system and to achieve resonance with the preferred
imaging frequencies of about 0.1-15 MHz, it may be
convenient to employ microbubbles and microparticles
having an average size of 0.1-10 ~m, e.g. 1-7 ~m; the
use of microparticles of average size 1-4 ~m to generate -
~microbubbles with an average size of 4-7 ~m is generally
advantageous. Substantially larger bubbles and
particles, e.g. with average sizes up to 500 ~m, may
however be useful in other applications, for example
gastrointestinal imaging.
The following non-limitative Examples serve to
illustrate the invention:-
.
.~
~'093/13809 PC~/EP93/O()O~X
2~2 ~70'~
Examples 1-3
D-(+)-~alactose~Starch Mixtures
D-(+)-Galactose (Merc}:) ir. the amoun.s stated in
Table I was mixed with starch (Reppal PSM 70, Reppe
Glykos, Sweden) in the stated amounts and ground for lO
minutes under aseptic conditions in a stainless steel
ball-~ill having a 50 ml grinding cup and 3x20 mm balls
(Retsch centrifugal ball-r.ill, Sl).
Table I
E~:am~le I A~,oun~. o: A~oun of .
No.ri~ )-qalacroc- s~arcrl
3 .1 ( a j i
i
; 2 ~ .2
. 1 3 3.2 0.8
Example 4
~-( )-Galactose~lron O~:ide l~i~:ture
Commercially available D-(-)-galactose (~.Og) was
mixed with commerciall~ available maqnetite (Fe3Q4 -
5.0~) and ground in a ball-rill as described in E~amples
1-3.
Example 5
D-(+)-Galactose~Starch~Palmitic Acid ~ixture
D-(+)-Galactose (5.0g! wa~ dissolved in distill.ed
water (7.1~) at 60~C and ~l~ed with starch (~`~.0~)
dissolved in distille(l wat.e~ \.0~) at ~ C. E'alr~itic
acid (0.0~9) was dis.sc).lvecl i~ ; ethanol (l.~) ancl
added with stirrincl to the car~)clhvc~rate solution at
60~C. ~he solvents wer~ evapor;lted under reduced
pressure (lO torr, GO~C) and the resulting solid ~i~ture
was dried in a desiccator overniaht prior to ~eing
ground in a ball-.~i'l as ;iescri~ed ln t~:a~ples ~
WO93/13809 PCT/EP93/0002X
~ .~ 2
C .
Examples 6 and 7
D-(+)-Galactose/Dextran Mixtures
D-(+)-Galactose in the amounts s ated in Table II
was dissolved in the stated weights of purified water at
50C. Dextran (molecular t~eight ca. 20 00~) in the
stated amounts was similarly dissolved in the stated
weights of purified water at 50 C. The solutions were
sterile filtered (0.22 ~m filter) and mixed with
stirring whereafter the solvent was evaporated under
reduced ~ressure (10 torr 40~C~. The resulting solid
mi~:tures l.iere dried in a ciesiccato~ overrliqh~ and then
grounc in a ball-mill as àescribel in r~:amp~es 1-3.
Table II
Exalr~?le ~r.ou~.~ o' Weight o~ a~er j A:n~a~ 9:~ cr water
No. D~ a_a^tos~ useà to à~ss~ e cex _a~ s- I to àissolve
~: ~ 9 ) D- ( ~ ) -ga 1 actos~ ! ~ 9 ~ ¦ dex ra~.
i l
6 ,.~ 1~ 14.~
, 2. 7.2 1 7.~ 21~ .
.
Example 8
Echo~enicit~ in vitro
10 ~1 of propylene glycol mi~ed ~ith 90 ml of 5%
de~:trose in water was used as a carrier liquid for
determining the echogenicity of products according to
the E~amples. l.Og of each product was dispersed in 3.0
ml of the carrier liquid and sha~en for 15 seconds. The
resultinc~ mi~:ture was added to S2 ml of 5~ human serum
albumin infusion solution in the measurement cell and
the acoustic effects of the prod~lcts t~ere investigated
by measu~in-~ the acoustic transmission throu~h the
samples usin~ a 5 MH7 broadband transducer in a pulse-
reflection technique. The temperature in the
measurement cell t:as sta~ilised tc 3~ C and circulation
~4093/13809 P~r/Er93/0002X
~ 1~7 7 ~ ~;
of the liquid was ~aintained by means o-: stirring at a
constant rate. Ultrasound transmission through the
samples was measured as a function of time over a
duration of 390 seconds. Results were normalized to
measurements on a reference consisting of 55 ml of 5
human serum albumin infusion solution.
The products of the Examples generally showed
higher echogenicit~ than the reference.
